Interest in cGMP as a second messenger has dramatically escalated in recent years. The list of cGMP actions in mammalian tissues is now quite large, and it is growing. The cGMP-dependent protein kinase (PKG) is a major intracellular receptor for cGMP. Elucidation of the physiological regulation of PKG is the long-term objective of this investigation. In addition to the classical roles ascribed to PKG in mediating effects of natriuretic peptides, nitric oxide or guanylins on airway and vascular smooth muscle relaxation, inhibition of platelet aggregation, and neutrophil degranulation, PKG may also mediate the cGMP-dependent effects of these and other agents on gene expression, chloride transport in intestine and kidney, heart contractility, water transport through the vascular endothelium, bone resorption, melanogenesis in skin, long-term nerve depression and opioid effects. PKG activation is believed to account for many of the pharmacological actions of medications such as "PDE inhibitors" (e.g., caffeine, papaverine) and nitrovasodilators (e.g., nitroglycerin) which are used for relief of chest pain, asthma, male impotence, and high blood pressure. PKG may also mediate the secretory diarrhea caused by certain bacterial enterotoxins. The importance of PKG has recently been enhanced by the realization that some effects of cAMP are mediated by cross-activation of PKG. The mechanism of dimerizaiton of PKG-I-alpha and PKG-I-beta will be studied using mutagenesis and proteolysis. Native and mutant PKG-I- alpha and PKG-I-beta will be utilized to define functional elements of the autoinhibitory domain and to study autophosphorylation. The autophosphorylation site(s) responsible for activation of each isoform will be identified. Conformational changes associated with cGMP binding and autophosphorylation will be measured using small angle X-ray scattering, gel filtration and native gel electrophoresis. PKG will be used as a model for other serine/threonine- and tyrosine-specific protein kinases that are activated by both ligand-binding (e.g., cyclic nucleotides Ca2+/calmodulin, insulin, growth factors) and autophosphorylation by determining whether or not activation by cGMP- binding or autophosphorylation produces the same enzyme conformation. The molecular mechanism of the activation processes will be examined. Native gel electrophoresis, which separates the different autophosphorylated species of the PKGs, and liquid chromatography-mass spectrometry will be used to determine if these species are present in intact tissues. Physiological regulation of PKG protein and mRNA levels will be explored. Results of these investigations will address major aspects of cGMP signaling through PKG.